The induction of apoptosis by chemotherapy drugs involves the activation of intracellular proteases, and abrogation of protease cascades can lead to drug resistance. Previous studies have revealed an important role for caspase proteases in apoptosis execution. However, little is known about the involvement of noncaspase proteases in drug-induced cell death. The identification and characterization of new death effector proteases, and the pathways they mediate, will provide new targets for future anticancer agents, and define new biomarkers of chemosensitivity or resistance. Therefore, our broad, long-term objective is to understand the role and mechanism of action of noncaspase proteases, primarily members of the cathepsin protease family, in chemotherapy-induced cell death. Recent studies suggest an important role for cathepsins in apoptotic execution. We have shown that treatment of cells with VP-16 induces processing of procathepsin D to active subunits. Inhibition of cathepsins substantially inhibited drug-induced cell death. Others have shown that downregulation of cathepsin D leads to delayed apoptosis. A potential mechanism of cathepsin D action is indicated by our finding that cathepsin D mediates cleavage of procaspase-8. Thus, we hypothesize that cathepsin D protease plays a critical role in chemotherapy-induced apoptotic execution, and acts to regulate the activation of caspase proteases. To test this hypothesis, we will assay cathepsin D activation in VP-16-treated cells, and employ fluorescent derivatives of cathepsin D to investigate potential drug-induced subcellular redistribution. Antisense and gene inactivation strategies will be used to downregulate cathepsin D, and the impact on multiple parameters of drug-induced apoptosis will be assessed, including caspase activation and loss of viability. Inhibition of apoptosis events will be used to further establish the position of cathepsin D in apoptotic pathways. Moreover, we will determine: i) whether cathepsin D directly cleaves procaspase-8, ii) whether cathepsin D activates or inactivates caspase-8, and iii) the location of the cathepsin D-mediated cleavage site. Finally, mutants of procaspase-8 will be generated and expressed in cells to determine whether the effects of cathepsin D are mediated through cleavage of procaspase-8. Together, these studies will delineate the importance of cathepsin D in VP- 16-induced cell death, and define the mechanism of action of this protease.